The present invention relates to methods and devices for determination of a leukocyte count and/or leukocyte subset count in biological fluids.
The tests for white blood cell, WBC (leukocyte) count and leukocyte subsets count are widely used in clinical practice. In general, laboratory methods for quantification of WBC or WBC subsets are based on the use of automated cell counting instruments and require 3-5 mL of venous blood samples, which are first diluted and then cells of different sizes and shapes are counted in a flow chamber[1]. Instruments which are utilized for automated cell counting are complex and costly, and only available in very few central hospitals and other larger laboratories. In addition, they require professionally trained personnel to operate the instruments.
At present, there is an increasing demand to reduce the turnaround time of test results, and new point-of-care testing (POCT) technologies are being developed for rapid diagnosis at or near patients' bedside. POCT devices are portable and user-friendly to operate, and, therefore, are of great value in both developing and developed countries, where access to automated cell counters is restricted, for instance in rural clinics or general practices.
Rapid measurements of leukocyte and their subsets are important in many clinical situations. They can be useful when physicians need to make decisions regarding the initiation (or monitoring) of treatment when the patient is still at clinical setting. The ability of POCT to provide results within minutes allows physicians to do it and use the results right away.
In hematology testing, there are three types of technology to support POCT: small bench-top analyzers, hand-held devices and manual tests. The bench-top systems are often smaller versions of laboratory analyzers, providing a full blood count (FBC) with red cell indices and either a five-part white cell differential or a partial three-part differential. Bench-top analyzers, however, are not useful at the patient's bedside and are not truly POCT devices because they are designed for use in clinics or small laboratories. The hand-held test devices and manual methods include measurement of hemoglobin (Hb) concentration, WBC and platelet count, detection of malaria and enumeration of CD4+ T-lymphocytes for human-immunodeficiency-virus (HIV) diagnosis and treatment monitoring[2; 3; 4].
Clinicians routinely use WBC and differentials (subsets) as biomarkers for acute infection/inflammation in various clinical settings from primary to critical care. An increased WBC count occurs in infection, allergy, systemic illness, inflammation, tissue injury, and leukemia. A low WBC count may occur in viral infections, immunodeficiency states, post chemotherapy, acute leukemia. For example, patients who are on chemotherapy need to check their WBC count frequently to ensure that they are eligible for the next treatment.
An increased or decreased total white blood cell count (WBC) could be due to abnormal bone marrow pathology[5]. Leukocytosis with an associated neutrophilia or lymphocytosis could infer the presence of a microbial or viral infection[3]. Leukocytosis is also a prognostic marker of patients who are at a higher risk of hospital mortality[6; 7] and identifies patients at increased risk for excessive bleeding[8]. Thus, clinicians can use the WBC biomarker to improve risk prognostication and identify patients in need of immediate treatment and a closer follow-up[9].
At present, there are several commercially available methods for the WBC count that can be used as POCT tests: 1) HemoCue WBC (Angelholm) [10], 2) the Chempaq XBC analyser (Chempaq A/S; Hirsemarken 1B, Farum, Denmark) [11], 3) PortaWBC™ (PortaScience, Moorestown, N.J.), and 4) the traditional procedure for the total and differential WBC count by manual microscopy[12].
The HemoCue WBC device measures total WBC without giving any differential. It consists of a microscopic image detector, a cuvette holder and an LCD display unit. The method is based on drawing peripheral capillary blood or venous blood into a plastic cuvette containing a reagent where the red cells are haemolysed and the nuclei of the white cells stained by methylene blue. Then, an image is captured, and the image analysis program counts WBC.
The Chempaq XBC hematology analyzers comprise a disposable cartridge and an instrument, and use impedance cell counting and measurement of Hb by a spectrophotometric method on 20 μl of blood. Thus, the aforementioned two instruments use rather complex instrumentation.
The PortaWBC™ method for quantitatively measuring white blood cell count involves capture of white blood cells from a fluid sample by a retainer that has a dye substrate immobilized therein, washing, and reading the result of a color reaction in which an ester which is present on the white blood cells cleaves a chromogenic substrate which produces a water insoluble dye. The signal is read using a glucose-like meter which measures the sample reflectance.
The total WBC count is obtained by lysing red blood cells in 2% acetic acid solution and counting WBCs in the hemacytometer chamber. For the differential WBC count, a stained smear is examined under microscope in order to determine the percentage of each type of leukocyte present. Stains for preparing whole blood smears are available from numerous manufacturers. Standard manual cell counting methods are time consuming and subjective. It is a common occurrence to obtain cell counts with wide inconsistencies in total cell counts. Likewise, the traditional procedure for the differential WBC count has a poor statistical reliability. In addition to that, it is time consuming, requires experience to make technically adequate smears consistently, and therefore is one of the most expensive routine tests in the clinical hematology laboratory[13]. Although the latter two methods are simpler and use less expensive equipment than the first two methods, they are labor-intensive and time-consuming. In addition, the PortaWBC™ method does not allow for determination of leukocyte subsets such as neutrophils, eosinophils, basophils, lymphocytes, monocytes, macrophages, dendritic cells, and granulocytes.
Very important parameters which are measured for diagnosis of HIV infection and monitoring of HIV patients are CD4+ T-lymphocyte levels for adults and CD4 percentage for pediatric patients.
HIV infection is one of the major problems in public health. It is estimated that more than 1.1 million HIV-infected persons are living in the United States and roughly 33.3 million people are living with HIV worldwide. In the recent years much importance is being given to a wider HIV screening and identification of infected individuals for implementation of intervention strategies. HIV testing has gained immense therapeutic relevance: starting the highly active antiretroviral therapy (HAART) early may improve quality of life and considerably prolong life. Treatment with HAART has dramatically improved survival rates in HIV since its introduction in 1995.
Individuals with HIV infection and AIDS exhibit abnormalities of the immune system, reflected primarily in their CD4 T lymphocytes, which are targeted by the virus. In adults, the evaluation of CD4 levels provides an important assessment of immunologic competency and has proven to be the most important test for HIV progression. Results from the measurement of CD4 lymphocyte levels provide information that guides therapy and predicts disease outcome. For example, new US guidelines favor antiretroviral therapy for patients at 350-500 CD4 cells/μL. New WHO recommendations are to start HAART for HIV patients with CD4 cell counts at or below 350 cells/μL, instead of a CD4 cell count of 200 cells/μL, the threshold which the WHO recommended in its 2006 guidelines. Normally, T lymphocytes (CD4 and CD8) account for 60-90% of all lymphocytes. Their numbers are about 1,600 cells/μl, with CD4 cells accounting for approximately 1,000 and CD8 cells approximately 500. CD4 cells are the main target of HIV and the number of CD4 cells will gradually decrease during HIV infection (50-100 cells/μl per year). HIV-positive individuals who are successfully treated with HAART demonstrate an increase in the CD4 cell count.
In pediatric patients, the high variability of absolute CD4+ T count occurs within the first 5 years of age. In addition, incurrent illnesses may affect CD4+ counts and the “normal” reference ranges for CD4 T+ cell absolute counts in African children differ from those reported for populations in Europe and North America[14; 15]. Therefore, in pediatric patients aged less than 5 years, it is strongly recommended that percentage of CD4+ T cells is determined within the total lymphocyte population (% CD4/ly)[16; 17]. If available, the percentage of CD4+ T cells is utilized to establish the level of immunodeficiency and make decisions when to start cotrimoxazole (CTX) prophylaxis and/or ART in all HIV infected children less than 5 years of age[18].
Relative to adults, very high absolute lymphocyte counts are seen in neonates (6,500±2,200 (SD) lymphocytes/μL) with a gradual decrease to near adult levels in children greater than 5 years of age (1,900±550 (SD) lymphocytes/μL)[19]. For classification of the HIV-associated immunodeficiency, the following % CD4/ly values are considered: mild immunodeficiency is suggested if % CD4/ly is 30-35% in infants, 25-30% in children aged 12-35 months, and 20-25% in children between 36-59 months of age. Severe immunodeficiency occurs when the % CD4/ly values in the age groups mentioned above drop below 25%, 20%, and 15%, respectively[18] World Health Organization (WHO) recommends initiation of CTX prophylaxis in all HIV-exposed children under the age of 1 year irrespective of their % CD4/ly measurement, particularly, in resource limited settings where infant HIV status may not be established until the age of 18 months due to the lack of proper technology[18]. In settings where % CD4/ly measurements are available, WHO recommends to initiate CTX prophylaxis in the 1-4 year age group if % CD4+/ly is less than 25%. The initiation of ART is determined by clinical presentation of disease and % CD4/ly measurements. ART is indicated for all HIV-infected individuals with an AIDS defining illness[18]. In pediatric patients <5 years of age with non-AIDS defining illness, ART is initiated at severe level of immune deficiency as determined by % CD4/ly[18].
Strategies to Assess Lymphocytes. Lymphocyte subsets are typically measured by immuno-fluorescent labeling of cells with fluorochromes conjugated to specific monoclonal antibodies and quantifying the proportion of specifically labeled cells by flow cytometry. Manual alternatives to flow cytometry are also available to quantify CD4 cells. They are simple light or fluorescence microscopy methods that just require cell counting.
Flow Cytometry. In flow cytometry, specific monoclonal antibodies made against the specific CD antigens present on the cells are labeled with fluorescent dyes. The labeled monoclonals are allowed to react with the mononuclear cells (lymphocytes and monocytes), and the cells that react can be classified by the flow cytometer into subpopulations depending on which monoclonals are bound. Flow cytometry generally gives the percentage of CD4+ or CD8+ cells. To obtain absolute cell counts, dual and single platform technologies are used. A dual platform technology employs a flow cytometer and a hematology analyzer. CD4 absolute count using dual platform approach is a product of three measurements: the white blood cell count, the percentage of white blood cells counts that are lymphocytes (differential), and the percentage of lymphocytes that are CD4 cells (determined by flow cytometry). If a single platform technology is used, absolute counts of lymphocyte subsets are measured in a single tube by a single instrument. Usually it is accomplished by spiking a fixed volume of sample with a known number of fluorescent beads (bead-based systems) or by precisely recording the volume of the sample analyzed. Recent recommendations suggest that single platform technology should be the gold standard for the CD4 absolute count.
Several varieties of flow cytometers are available, with the FACSCalibur (Becton Dickinson) and EPICS XL (Beckman Coulter) being the most popular. These instruments offer high sample throughput, workflow management through automation, and simple software applications. Both instruments can detect four colors and measure relative cell size and cellular complexity. The systems are designed to use whole blood, collected in liquid EDTA. Besides using the traditional flow cytometers (open platforms that can employ dual or single platform technology), the simplified dedicated platforms are developed for CD4+ T-cell counts. The commercially available dedicated platforms include FACScount (Becton Dickinson), CyFLow Counter (Partec), and Guava Auto CD4/CD8% (Millipore/Merck). The dedicated platforms allow CD4+ T-cell counting with reduced technical complexity. It produces absolute CD4 counts and a CD4/CD8 ratio without requiring an external computer. The system uses whole blood, eliminates the need for lysis and wash steps, and has a unique software algorithm that automatically identifies the lymphocyte populations of interests. However, the instrument costs about $25,000, with each assay costing $3-20 depending on volume of tests performed in the laboratory.
Flow cytometry, even though the “gold standard” reference method for determining lymphocyte subpopulations, has several disadvantages: the method requires an expensive instrument ($25,000-90,000), an expensive service contract, and well-trained personnel. Highly trained personnel and costly equipment make it difficult for small laboratories or those in developing countries to routinely provide this testing.
Manual Methods to Quantify Lymphocytes. Manual alternatives to flow cytometry available on the market are: the Cyto-Spheres (Coulter Corporation, USA) and the Dynabeads (Dynal AS, Norway). The Dynal T4 kit (the Dynabeads) is used to manually count CD4 cells in a cell counting chamber under a microscope. This method measures CD4 absolute count; no lymphocyte percentages can be determined. It requires an epifluorescent microscope (recommended), although it can be performed with only a light microscope; a hemacytometer, a vortex, a tube rocker, a timer, and a magnet. Magnetic beads are coated with monoclonal antibodies as a solid phase to isolate CD4 and CD8 cells from whole blood, whereas CD4-positive monocytes are pre-depleted using CD14 magnetic beads. After isolation of CD4 cells, the cells are lysed, stained, and counted. Blood samples should be fresh, preferably not older than 24 hours. The Coulter Manual CD4 Count Kit (cytospheres method) requires a light microscope, timer, and a hemacytometer and measures CD4 absolute counts (no percentages) from whole blood collected in EDTA tubes [20]. Antibody-coated latex particles are used to bind CD4 cells resulting in a “rosette” of latex beads around each CD4 cell; the rosette is readily recognized by light microscopy. A monocyte blocking reagent minimizes the interference from monocytes that contain CD4 antigens because they can be recognized during CD4 cell counting. Blood should be tested within 6 hours of collection and should not be refrigerated. Both manual assays cost between $5-6 per test and are designed to operate with low sample throughput in resource-limited laboratories. However, the manual methods are labor-intensive; require many manual steps, and an experienced microscopist. Large intra- and inter-operator variations are the norm rather than the exception and these methods have been proven very difficult to implement in most settings.
New CD4 Technologies. Recent development of CD4 technologies is concentrated on creating an accurate point-of-care (POC) assay that could be used for CD4 testing in resource-limited countries in any health care setting or in the field. Inverness Medical (now Alere) has a PIMA CD4 test based on static image analysis and counting principles. A disposable cartridge containing dried reagents and a portable analyzer is used. The analyzer is easy to operate: no extensive training is required. The cost of analyzer is $5,500; an estimated cost per test is $6. The assay is already available in select markets. Daktari Diagnostics is developing a microfluidic-based system to capture CD4 cells and to count cells using simple electrical impedance measured by a small portable device. The estimated device cost is $800 and the single test cost is estimated to be $8. mBio Diagnostics, a division of the Precision Photonics Corporation has a new patent-protected CD4 count technology that uses an integrated fluidic cartridge and a portable fluorescence imaging device. Zyomyx is another company involved in the development of a POC CD4 test: Zyomyx CD4 counter is similar to a thermometer where CD4 cell stacking height highly correlates with CD4 count in the blood sample. The major advantage of this assay is that it does not require any instrument for reading the results. The estimated cost of the assay is $6-7, however, this assay is still under development. A different new CD4 technology developed by Burnet Institute in Australia is based on the measurement of CD4 protein on T-cells, rather than measuring CD4 cells using a lateral flow assay. The test, which is expected to cost about $2, is semi-quantitative: it will be able to determine, whether a patient's CD4 count is above or below a threshold of 350 CD4 cells/μL, but it will not give full quantitative results. The reader of the test is expected to cost about $1,200. The above mentioned POC tests are promising, however, they still need to be evaluated in the clinical trials: peer reviewed published data is available only for PIMA CD4 test. All other tests still need to stand up to robust assessment.
Current flow cytometric methods to measure CD4 cells are expensive, require significant technical expertise, the instrumentation has high service demands, and these methods are difficult to support in resource-limited countries. The aforementioned manual CD4 tests have their advantages and limitations. Indeed, Dynabeads (Dynal Biosciences) and Cytospheres (Coulter), methods are multi-step assays and require a microscope for counting targeted cells, what leads to increasing of the assay cost. In addition, these manual methods are laboratory-based and their procedures are not conducive for point-of-care workers such as nurses, counselors, and physicians. Moreover, currently there are no manual methods on the market that provide % CD4/ly measurement. Thus, the rationale is to develop and validate a truly point-of-care CD4 method that can be used by non-laboratory personnel in a wide variety of health care settings and in the field to provide anti-retroviral treatment decisions immediately and without the need for infected persons to return for results. Serious limitations of the aforementioned methods hinder their wide use in clinical practice. Therefore, the development of an alternative manual assay that is free from the above drawbacks is of utmost relevance.